Biochar has shown great potential for immobilizing organic contaminants in soil. In this study, pentachlorobenzene (PeCB), 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB), and 1,2,4-trichlorobenzene (1,2,4-TCB) artificially spiked soil was amended with wheat straw biochar at 0.1%, 0.5%, 1%, and 2% application rates, respectively. The sorption, dissipation, and bioavailability of chlorobenzenes (CBs) in soil were investigated. The sorption of PeCB by biochar was significantly higher than that of its sorption by both biochar-amended and unamended soil (p < 0.05). The dissipation and volatilization of CBs from biochar-amended soil significantly decreased relative to unamended soil (p < 0.05). Bioavailability of CBs, expressed as butanol extraction efficiency and earthworm (Eisenia fetida) bioaccumulation factor, significantly decreased with increasing aging time and biochar application rate. The effect of biochar content in soil on the bioavailability of CBs was more pronounced for 1,2,4-TCB relative to other CBs. This study suggested that wheat straw biochar, even at low application rates, could effectively immobilize the semivolatile CBs in soil and thus reduce their volatilization and bioavailability.